Hemoglobin, a respiratory heme protein, is commonly present in the blood of vertebrates. Elemental oxygen molecules bind to the iron atoms of the hemoglobin in the lungs where oxygen is abundant, and are released later in tissues that need oxygen. Depending on the oxidation states of heme iron, two forms of hemoglobin, ferrous hemoglobin (Fe2+) and ferric (or met) hemoglobin (Fe3+) are found naturally. Both forms of protein can bind with various ligands in solution. A characteristic visible spectrum is generally associated with each species of liganded hemoglobin.
The determination of total hemoglobin is indicative of the oxygen-carrying capacity of whole blood. An ability to measure hemoglobin (Hgb) in blood samples is an essential part of diagnostic analysis and is also important for monitoring responsiveness to therapies directed towards diseases which affect hemoglobin and to therapies which are directed towards other diseases but which may have adverse side effects on the hemoglobin level.
Leukocytes in the peripheral blood of normal subjects consist of five types, i.e., lymphocytes, monocytes, neutrophils, cosinophils and basophils. The latter three types of leukocytes are collectively referred to as granulocytes. Different types of leukocytes have different biological functionalities. Counting and differentiating different types of leukocytes in a blood sample provides valuable information for clinical diagnosis.
The counting and classification of leukocytes has most commonly been conducted by automatic hematology analyzers. They employ a hemoloytic reagent to lyse erythrocytes and produce a sample only containing leukocytes. The sample mixture then is analyzed by impedance method. It is important that the lyse reagent not damage the leukocytes. A more sophisticated apparatus has been developed that counts different types of leukocytes (differential counting) including lymphoid (lymphocyte) and myeloid (monocyte and granulocyte) populations. Ideally, one would like to be able to accomplish multiple diagnostic analyses such as hemoglobin measurement and counting the number of leukocytes or differential counting of leukocyte subpopulations in a single automated step.
Hemoglobin count, in g/dL, can be accurately obtained using automated hematology analyzers. Most analyzers use potassium cyanide (KCN) as the key component in lysing agent, due to the extremely stable absorption peak at 540 nm for cyanomethemoglobin. A single-wavelength light absorption measurement is designed for these instruments, in order to give the absorbance (A540) of cyanomethemoglobin solution, and subsequently, the correct count of hemoglobin (HGB) in whole blood or control samples.
The cyanide hemoglobin method has been recommended as a standard by the International Committee for Standardization in Hematology. Modification of this method has led to its wide usage in clinical laboratories. In this method, the iron ion of heme group in all forms of hemoglobin of the red cells are oxidized to methemoglobin by potassium ferricyanide. The methemoglobin is then complexed with cyanide anion, which has a very high affinity to iron ion of the heme group, to form a cyanmethemoglobin chromogen. This extremely stable chromogen has a maximum absorption at 540 nm, which is measured manually by UV spectrometry.
Despite the stable chromogens formed by the standard cyanmethemoglobin method and its modified automatic methods, It is important to develop a cyanide free reagent because the potassium cyanide reagent has caused raised environmental concern with waste disposal. In last ten years, a effort has been given to develop automated hemoglobin analysis methods without utilizing cyanide.